Skin cells must respond to DNA damage caused by both environmental exposure as well as therapeutic agents. One response, DNA repair, is the process by which cells recognize, remove and replace lesions in their genomes. Defects in DNA repair underlie several heritable diseases that predispose individuals to skin cancer, photosensitivity and developmental defects. Although impaired repair of random DNA damage is deleterious, targeted DNA damage and selective inhibition of its repair in certain genes can be desirable to achieve a therapeutic effect. Triplex-forming oligonucleotides (TFOs) that target a DNA damaging agent to a specific nucleotide sequence have emerged as a promising approach for gene therapy. TFOs create structurally unconventional types of DNA damage that can provide insight into the mechanisms of normal DNA repair, that may actually mimic naturally occurring macromolecular interactions that are involved in diseases of the skin, and that have potential to develop into a practical form of cutaneous gene therapy. The overall objective of the proposed project is to understand and manipulate how TFOs target psoralen photoadducts to the interstitial collagenase gene in skin derived cells, and to identify the molecular basis for repair of this unusual macromolecular damage. The hypothesis is that TFO and chromatin structures actively modulate both delivery and repair of a unique form of macromolecular DNA damage, and that novel cellular mechanisms exist to repair this type of damage. To test this hypothesis, the specific aims of the project are: 1) To assess the role of TFO structure in delivery and repair of psoralen photoadduct damage in genomic targets. TFOs with modified backbones will be assessed for their ability to target psoralen photoadducts and mediate their repair in the interstitial collagenase gene; 2) To determine the role of chromatin structure and gene activity in TFO-targeted DNA damage and repair. Exogenous agents that generally or focally disrupt chromatin structure or that alter transcription will be examined for the ability to affect accessibility of TFO target sites to damage and repair; 3) To determine the mechanism of nucleotide excision repair in removal of psoralen adducts targeted by TFOs. Structural TFO variants and human cell lines defective in DNA repair will be used to dissect the role of nucleotide excision repair in processing psoralen photoadducts targeted by different TFOs; 4) To assess the role of cellular helicases in repairing TFO lesions. Cell lines with different helicase activities or deficiencies will be used to determine if these enzymes participate in processing of TFO-mediated damage. At the conclusion of the project period, the proposed work should contribute to our fundamental knowledge of DNA repair mechanisms as well as provide basic principles to guide therapeutic applications of TFOs by manipulating DNA damage and repair.